1. DNA Replication
Chapter 25

2. DNA Polymerase (E. coli ex)
Catalyzes synth of new DNA strand
d(NMP)n + dNTP  d(NMP)n PPi
3’ –OH of newly synth’d strand attacks first phosphate of incoming dNTP
Rxn thermodynamically favorable
Why??

3. DNA Polymerase – cont’d
Noncovalent stabilizing forces impt
REMEMBER??
Base stacking  hydrophobic interactions
Base pairing  multiple H-bonds between duplex strands
As length of helix incr’d, # of these forces incr’d  incr’d stabilization

4. DNA Polymerase – cont’d
Can only add nucleotides to pre- existing strand
So problematic at beginning of repl’n
Problem solved by synth of ….

5. DNA Polymerase – cont’d
Primers (25-5)
Synth’d by specialized enzymes
Nucleic acid segments complementary to template
Often RNA
Have free 3’ –OH that can attack dNTP

6. DNA Polymerase – cont’d
Once DNA polymerase begins synth of DNA chain, can dissociate OR can continue along template adding more nucleotides to growing chain
Rate of synth DNA depends on ability of enz to continue w/out falling off
Processivity

7. DNA Polymerase – Accuracy
Enz must ACCURATELY add correct nucleotide to growing chain
E. coli accuracy ~ 1 mistake per 109 – nucleotides added
Geometry of enz active site matches geom. of correct base pairs (25-6)
A=T, G=C fit
Other pairings don’t fit

8. Fig.25-6

9. Accuracy – cont’d Enz has “back-up” proofreading ability
Its conform’n allows recognition of improper pairing
Has ability to cleave improperly paired bases (25-7)
Called 3’  5’ exonuclease activity
Enz won’t proceed to next base if previous base improper
Then catalyzes add’n of proper base
Increases accuracy of polymerization 102 – 103 X
Note: cell has other enz’s/mech’s to find/repair mistakes (mutations) after new helix synth’d w/ repl’n

10. Fig.25-7

11. 3 E. coli DNA Polymerases (Table 25-1)
I -- impt to polymerase activity
Slow (adds nucleotides/sec)
Has 2 proofreading functions
Only 1 subunit
II – impt to DNA repair
Less polymerization activity
Several subunits

13. 3 DNA Polymerases -- cont’d
III – principle repl’n enz
Much faster than polymerase I (adds nucleotides/sec)
Many subunits (Table 25-2) each w/ partic function
Encircles DNA; slides along helix (25-10)
One subunit “clamps” helix  better processivity

14. Fig.25-10

15. Replisome Many other enz’s/prot’s necessary for repl’n (Table 25-3)
Complex together  replisome
Helicases – sep strands
Topoisomerases – relieve strain w/ sep’n
Binding proteins – keep parent strands from reannealing
Primases – synth primers
Ligases – seal backbone
What bonds hold nucleic acid backbone together?

16. Initiation – 1st Stage Repl’n
E. coli unique site = ori C (25-11)
3 adjoining 13-nucleotide consensus seq’s
Non-consensus “spacer” nucleotides
4 9-nucleotide consensus seq’s spaced apart
Consensus seq’s contain nucleotides in partic seq common to many species

17. Initiation – cont’d At ori C (at 4 9- ‘tide seq area) (25-12)
~20 DnaA mol’s (proteins) bind
Requires ATP
 nucleosome- like structure

18. Initiation – cont’d  Unwinding of helix (at 3 13- ‘tide seq area)
~13 nucleotides participate in unwinding
Requires ATP
Requires HU (histone-like protein)

19. Initiation – cont’d Unwound helix is stabilized
Requires DnaB, DnaC (proteins)
These bind to open helix
DnaB also acts as helicase
Unwinds DNA helix by 1000’s of bp’s

20. Initiation – cont’d Result:
Nucleotide bases now exposed for base pairing in semiconservative repl'n
What does semiconservative mean?
Yields 2 repl’n forks

21. Initiation – cont’d
Other impt repl’n factors at repl’n forks (Table 25-4)
SSB = Single Strand DNA Binding Protein
Stabilizes sep’d DNA strands
Prevents renaturation
DNA gyrase -- a topoisomerase
Relieves physical stress of unwinding
Note: in E. coli, repl’n is regulated ONLY @ initiation

22. Elongation Second stage of repl’n
Must synth both leading and lagging strands
REMEMBER: 1 parent strand 3'  5; its daughter can be synth'd 5'  3' easily. What about the other parent strand (runs 5'  3')??
Follows init’n w/ successful unwinding  repl’n fork, stabilized by prot’s
So have parent strands available as templates for base-pairing  2 daughter dbl helices

23. Elongation -- cont'd – Leading Strand
Simpler, more direct (25-13)
Primase (=DnaG) synthesizes primer
10-60 nucleotides
NOT deoxynucleotides
Short RNA segment
fork opening
Yields free 3’ –OH that will attack further dNTP’s

24. Leading Strand – cont’d
DNA polymerase III now associates
Catalyzes add’n of deoxy- nucleotides to 3’ –OH (25-5)

25. Leading Strand – cont’d
Elongation of leading strand keeps up w/ unwinding of repl’n fork
Gyrase/helicase unwind more DNA  further repl’n fork
SSB stabilizes single strand DNA til polymerase arrives
Synth continues 5’  3’ along daughter strand

26. Fig.25-13

27. Elongation -- cont'd – Lagging Strand
More complicated
REMEMBER: still need 5’  3’ synth, AND still need to have antiparallel strands.
Template strand here is 5’  3’
Can’t synth continuous daughter strand 5’  3’
Cell synth’s discontinuous DNA fragments (Okazaki fragments) that will be joined (25-13)
Must have several primers AND coordinated fork movement

28. Fig.25-13

29. Lagging Strand – cont’d
Lagging strand is looped next to leading strand (25-14)
DNA polymerase III complex of subunits catalyzes nucleic acid elongation on both strands simultaneously
Primosome = DnaB, DnaG (primase) held together w/ DNA polymerase III by other prot’s

30. Fig.25-14

31. Lagging Strand – cont’d
One subunit complex of DNA polymerase III moves along lagging fork in 3’  5’ direction (along parent)
Another subunit complex of polymerase III synth’s daughter strand along leading strand
At intervals, primase attaches to DnaB (helicase)
Here, primase catalyzes synth of primer (as on leading strand)
Also (once primer synth’d), primase directs “clamp” subunit of polymerase III to this site
This directs other polymerase III subunits to primer

32. Lagging Strand – cont’d
Now polymerase III catalytic subunits add deoxynucleotides to primer  Okazaki fragment
Book notes primosome moves 3’  5’ along daughter strand, but both primase & polymerase synthesize strands 5’  3’ along daughter

33. Fig.25-14

34. Lagging Strand – cont’d
Okazaki fragments must be joined
DNA polymerase I exonuclease cleaves RNA primer (25-15)
DNA polymerase I simultaneously synth’s deoxynucleotide fragment
10-60 nucleotides
Nicks between fragments

35. Lagging Strand – cont’d
DNA ligase seals nicks between fragments (25-16)
Catalyzes synth of phosphodiester bond
NADH impt (coordination role?)

36. Fig.25-16

37. Termination Repl'n has occurred bidirectionally @ 2 forks concurrently
E. coli genome is closed circular
So 2 repl'n forks will meet

38. Termination – cont’d Ter = seq of ~ 20 nucleotides (25-17)
Tus = prot's that bind Ter
When replisome encounters Ter-Tus
Replisome halted
Repl'n halted
Replisome complex dissociates

39. Termination – cont’d Result = 2 intertwined (catenated) circles
Topoisomerase IV nicks chains
One chain winds through other
2 Complete genomes sep'd

40. Eukaryotic DNA Replication
Repl'n mechanism & replisome structures similar to prokaryotes, BUT:
DNA more complex
Not all is coding for peptides
Chromatin packaging more complex
REMEMBER: nucleosomes, 30 nm fibers, nuclear scaffold, etc.
No single origination pt for repl'n
Many forks develop
 Simultaneous repl'ns bidirectionally
Forks move more slowly than in E. coli
But efficient because more forks

41. Eukaryotic DNA Replication
Repl'n enzymes not yet fully understood
DNA polymerase a
In nucleus
Has subunit w/ primase activity
May be impt to lagging strand synth
DNA polymerase d
Assoc'd w/ a
Impt to attaching enz to nucleic acid chain
Has 3'  5' exonuclease ability (proofreading)
DNA polymerase e
Impt in repair

42. Eukaryotic DNA Replication
Replisome proteins not yet fully understood
Found prot's similar to SSB prot's of E. coli
Termination seems to involve telomerases
Telomeres = ends of chromosomes

43. DNA Alterations
Need unaltered, correct nucleotide seq to code for correct aa's  correct peptides  correct proteins
Some changes acceptable
Some "wobble" in genetic code
Some DNA damage in mature cells can be fixed
DNA repair mechanisms avail for TT dimers (ex)
Have (more) other mature cells that can maintain homeostasis in organism
BUT -- if mispaired bases during repl'n  mutation in daughter cell (and her subsequent daughters)

44. Definitions Lesion = unrepaired DNA damage
Mammalian cell prod's > 104 lesions/day
Mutation = permanent change in nucleotide seq
Can be replicated during cell division
Results if DNA polymerase proofreading fails
May occur in unimpt region = Silent Mutation
Doesn't effect health of organism

45. Definitions – cont’d Mutation -- cont’d
May confer advantage to organism = Favorable
Rare
Impt in evolution
May be catastrophic to organism health
Correlations between mutations & carcinogenesis

46. DNA Repair Cell has biochem mech's to repair damage to DNA
Though 104 lesions/day, mutations < 1/1,000 bp's
If repair mech's defective  disease/dysfunction
Ex: xeroderma pigmentosum
UV light  DNA lesions
No repair mech
 Skin cancers
Repair mech ex: base excision repair
Takes advantage of complementarity of strands

47. Base Excision Repair N-Glycosylases Cleave N-glycosyl bonds
What parts of nucleic acid are joined by N- glycosyl bonds?
Several specific N-glycosylases
Each recognizes a common DNA lesion
Common -- bases altered by deamination events
Yields apurinic or apyrimidinic (AP) site

48. Excision Repair – cont’d
Uracil Glycosylase -- ex
Deamination of cytosine  uracil (improper)
Enz recognizes, cleaves ONLY U in DNA
Not U in RNA
Not T in DNA
 AP site on DNA (25-22)
Would this be apurinic or apyrimidinic?
Leaves behind sugar-phosphate of original nucleotide

49. Excision Repair – cont’d
Then other enz's (AP endonucleases) cleave several bases of mutated strand around AP site
Then DNA polymerase I catalyzes polymerization of proper nucleotides at site
Then DNA ligase seals nicks on sugar- phosphate backbone

50. Fig.25-22